Figure 8

Aβ oligomer induced neuronal CCEs are blocked with inhibitors of the PI3K/Akt/mTOR pathway. A. Cultured primary cortical neurons (21 DIV) were pretreated for 30 min with the PI3K inhibitor wortmannin (100 nM), an Akt inhibitor (100 nM) and the mTOR inhibitor rapamycin (1 μM), followed by exposure to 2.0 μg/ml of Aβ oligomers (AβO) for 24 hours in the presence of BrdU. Following fixation, cells were stained with specific antibodies against BrdU and MAP2. Quantification of the BrdU positive/MAP2 positive cells demonstrated a statistically significant decrease in the percentage of neurons positive for BrdU with all three inhibitors (p < 0.001 for AβO vs AβO+wortmannin, AβO+Akt inhibitor and AβO+rapamycin; one-way ANOVA with Tukey multiple comparison test for pair-wise comparisons; mean ± SEM; n = 4 independent treatments per group). B. Quantification of the loss of MAP2 positive processes upon treatment with AβO in the presence of the PI3K, Akt and mTOR inhibitors was determined via automated image processing and revealed a statistically significant increase in the number of MAP2 positive processes upon pre-treatment with the PI3K inhibitor (p = 0.0005; unpaired t test; mean ± SEM; n = 4 independent treatments), but not the Akt or mTOR inhibitors when compared to AβO treatment alone. C. Diagram outlining a potential pathway underlining the effects of Aβ oligomers on neuronal process retraction and CCEs. Our data suggest that Aβ oligomers activate the PI3K-Akt-mTOR signaling pathway. Activation of PI3K causes phosphorylation of Akt at Ser473, which in turn activates mTOR via phosphorylation at Ser2448. Activation of mTOR results in induction of cell proliferation by down regulation of 4E-BP1 via inhibitory phosphorylation at ser65. Interestingly, our data suggest that the dendritic alterations induced by Aβ oligomers is blocked via inhibition of PI3K via as yet to be identified downstream effectors.